This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. A window chamber system will allow visualization of cell co-localization with C-ECM components in a full thickness scaffold, in which mesenchymal stem cells (MSCs) are expected to co-localize with collagen I and endothelial progenitor cells (EPC) with collagen IV. The first goal will be to build a window chamber to visualize a full thickness C-ECM and its re-cellularization with EPCs and MSCs. Then, co-localization of seeded cells with C-ECM proteins (collagen I and IV) will be characterized. Lastly, the de-cellularization protocol will be optimized to preserve the C-ECM proteins with which cells were shown to co-localize, increasing the re-cellularization efficiency by increasing the attachment of cells to the C-ECM. Updated: The multi-photon microscopy will enable us to image at a greater depth than is currently possible with the confocal microscopes available in my lab. Second harmonic generation of collagen will be used to identify co-localization of mesenchymal cells with ECM components. External fluorescence will be imaged using fluorescent endothelial and mesenchymal stem cells and fluorescent antibodies specific to collagen IV laminin. The real-time imaging portion will be performed in our own lab using a confocal microscope.